生物化学技术

Since its discovery in 1957, coenzyme Q10 (CoQ10) has been extensively studied for its important role as a redox carrier in the mitochondrial electron transport chain and for its role as a potent antioxidant in animal tissues (1,2). In addition, several reports indicate that deficiencies in CoQ ...

It has been reported that in a system containing microsomal phospholipids, nicotinomide adenine dinucleotide phosphate (NADPH), Fe3+-ADP complex and cytochrome P-450 reductase (a reconstituted microsomal lipid peroxidation system), excited carbonyl species and singlet m ...

Measurement of thiobarbituric acid reacting substances (TBARS), diene derivatives, and hydroperoxides are probably the most widely used procedures for evaluation of free radical-mediated oxidation of biological or model systems containing polyunsaturated fatty acids ( ...

It is known that Hg and Cd causes Minamata and Itai-Itai (Auch-Auch) diseases because of the environmental pollution problems in some local areas of Japan. As a result, the high toxicity of compounds like Hg and Cd are now well-recognized. In addition, Pb, As, Cr(VI), Se, Be, Tl, Sn, etc. have also caused environm ...

Many compounds of great structural diversity possess varying degrees of estrogenic activity (Fig. 1). Estrogenic substances may contain a steroidal ring system as do, for instance, the ovarian hormones estradiol3 (E2), estrone (El), or the synthetic estrogens 17α-ethinylestradiol ...

In the past, the principal techniques of choice for analyzing tissue for various inorganic atomic species have been colometric and via atomic absorption spectrophotometry (1). Although both methods can be accurate and precise when performed in the same laboratory, there are disadvant ...

A photo-labile compound that is bioinactive but upon irradiation with light yields bioactive species, is called as “caged compound” (1) (see Scheme 1). Photolysis of caged compounds generating bioactive species, i.e., “uncaging,” has become a general method to produce a desired amount of bio ...

The potential of standard in vitro transcription reactions can be dramatically expanded, if chemically synthesized low-mol-wt compounds are used as building blocks in combination with standard nucleotide 5′ triphosphates (NTPs). Short oligonucleotides that terminate in gua ...

Human cytomegalovirus (HCMV) represents one of the most medically important human viruses and causes a wide spectrum of human diseases, including birth defects and mental retardation in newborns, common opportunistic infections in aquired immunodeficiency syndrom (AIDS) pat ...

The use of external guide sequence (EGS) in directing endogenous ribonuclease P (RNase P) for inhibition of viral propagation is described in this chapter, with an emphasis on chemically modified EGSs and their extracellular delivery. Targeting of the mRNA-encoding human cytomegalo ...

External guide sequences (EGSs) are oligonucleotides that consist of a sequence that is complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. Recent studies indicate that increasing the targeting activity of EGSs in directing human ...

Nucleic acid enzymes have emerged as a versatile technique for sequence-specific gene silencing in a wide range of cells. However, the question remains as to whether, for example, DNA enzymes and ribozymes are functional in animals. In this chapter, we describe two different rodent models of hu ...

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have varying silencing capacities, several different siRNAS typically must be screened to obtain a region that wi ...

Small interfering RNAs (siRNAs) have emerged as a powerful technique for sequence-specific gene silencing in a wide variety of organisms. However, the base composition of the siRNA sequence is not the only determinant of efficacy. Intrinsic factors related to mRNA structures are likely to ...

By introducing double-stranded RNAs (dsRNAs), it was shown that mRNAs that share sequences are destroyed, and that the translation step is severely downregulated (RNAi). This technique was demonstrated to be a very powerful tool for reverse genetics in Caenorhabditis elegans. However, ...

Post-transcriptional gene silencing is a powerful tool to reveal gene function. In this chapter, we have used gene-array technology to analyze changes in gene expression after specific targeting of the protein kinase Cα isoform with small interfering RNAs (siRNAs). Expression profil ...

RNA interference (RNAi) is a recently developed technique for gene silencing by introducing dsRNA into cells, and it is shown to work in mammalian cells when siRNAs are used. Several groups have developed vector-based siRNA expression systems that can induce RNAi in living cells. These vector ...

In mammalian cells, the RNA interence (RNAi) effect has been observed through expression of 21–23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. Here, we describe a facile polymerase chain reaction (PCR)-based strategy for rapid synthesis and evalu ...

The application of conventional enzymological methods to the study of hairpin and hammerhead ribozymes has led to valuable insights into the mechanisms by which these small RNAs catalyze phosphodiester cleavage and ligation reactions. Here, protocols are presented for measuring ...

RNA interference is the process that double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA mediated by 21- to 23-nt small interfering RNA (siRNA). Successful application of RNAi in functional genomics and proteomics, cancer gene therapy, and virus p ...